Therapeutic medicine for cystic fibrosis

ABSTRACT

The object of this invention is to provide a therapeutic medicine for cystic fibrosis. The therapeutic medicine for cystic fibrosis according to this invention contains an alginate lyase capable of lysing the alginate produced by strains of microorganisms of the genus Pseudomonas as the active ingredient.

TECHNICAL FIELD

This invention relates to a therapeutic medicine for cystic fibrosis.

BACKGROUND ART

In the white population, an inherited gene-defective disease known ascystic fibrosis is encountered with a frequency of 1/1000-2000. Thisdisease is caused by the defect of a gene coding for cystic fibrosistransmembrane conductance regulator (CFTR) protein and in the event ofbacterial infection to the respiratory organ with mucoid forms ofPseudomonas aeruginosa,. Mucous substances collect in the lungs to causean obstruction of airways leading to premature death. Though antibioticsand digestive enzymes are currently in use for the treatment of thisdisease, adequate therapeutic responses remain to be achieved as yet.

Recently (Richard C. Hubbard, et al.) have reported that symptomaticimprovements in cystic fibrosis may be expected if the DNA contained insaid mucus is lysed with a DNase [The New England Journal of Medicine326, 812-815, 1992]. However, the DNase is little capable of lysing themucus produced by bacteria of the genus Pseudomonas so that nosufficient efficacy can be expected.

Meanwhile, it is not deniable that the action of antibiotics anddigestive enzymes on Pseudomonas aeruginosa is compromised by thealginate which these microorganisms themselves produce.

DISCLOSURE OF INVENTION

The inventors of this invention did a great deal of research to developa therapeutically effective drug for cystic fibrosis. In the course oftheir exploration, the inventors discovered that the mucous substancesecreted by strains of Pseudomonas aeruginosa isolated from the lungs ofpatients with cystic fibrosis contained not only DNA but also alginatein an amount several-fold as great as the amount of DNA. Inspired by thethought that should this alginate be somehow decomposed the morbidity ofcystic fibrosis could be cured, the inventors did further research andhave found that a certain alginate lyase is effective for the treatmentof cystic fibrosis.

The object of this invention is to provide a drug suited for the therapyof cystic fibrosis.

In accordance with this invention there is provided a therapeuticmedicine for cystic fibrosis which comprises an alginate lyase capableof lysing the alginate produced by microorganisms of the genusPseudomonas as an active ingredient.

The alginate lyase that can be used as the active ingredient of thetherapeutic medicine of this invention is not restricted insofar as itis able to lyse the alginate produced by microorganisms of the genusPseudomonas. The preferred species of the-alginate lyase for thepurposes of this invention include the alginate lyase having anN-terminal amino acid sequence corresponding to SEQ ID NO: 1 presentedhereinafter and having the following physicochemical properties(hereinafter referred to as Al-I lyase) and the alginate lyase having anN-terminal amino acid sequence corresponding to SEQ ID NO: 2, which alsoappears hereinafter, and having the following physicochemical properties(hereinafter referred to as Al-III lyase), among others.

Physicochemical properties of Al-I lyase

(1) Activity: This enzyme lyses alginate to saccharides having anon-reducing end C₄ -C₅ double bond and ultimately to4-deoxy-5-ketouronic acid.

(2) Molecular weight: 60000

(3) Optimal pH: 8.0

(4) Stable pH: 6.0-8.0

(5) Optimal temperature: 70° C.

(6) Substrate specificity: Highly capable of lysing alginate of thebacterial origin

Physicochemical properties of Al-III lyase

(1) Activity: This enzyme lyses alginate to saccharides having anon-reducing end C₄ -C₅ double bond and ultimately to4-deoxy-5-ketouronic acid.

(2) Molecular weight: 38000

(3) Optimal pH: 8.0

(4) Stable pH: 6.0-8.0

(5) Optimal temperature: 70° C.

(6) Substrate specificity: Very highly capable of lysing alginate of thebacterial orgin.

The above physicochemical properties are now described in detail. Itshould be understood that the physicochemical properties of Al-I lyaseand Al-III lyase were determined using the purified enzyme samplesprepared in Reference Examples 1 and 2 which are presented hereinafter.

[Alginate lyase activity]

The activity of each alginate lyase was assayed according to theprinciple that the saccharides having a non-reducing end C₄ -C₅ doublebond as produced on lysis of alginate cause a specific increase inabsorbance at 235 nm.

Specifically, the enzymatic activity was determined as follows. First,1.0 ml of a 0.2% aqueous solution of the alginate, 0.5 ml of 200 mMTris-HCl buffer (pH 7.0) and 0.1 ml of the enzyme solution were mixedtogether and after the mixture was diluted with 0.4 ml of water to make2.0 ml, the reaction was carried out at 25° C. for 5 minutes. Thereaction was then stopped and the absorbance was measured at 235 nm. Theenzyme activity was expressed in the activity per mg of the enzyme withthe amount of enzyme increasing the absorbance at 235 nm by "1" in 1minute being taken as unity (U).

The substrate used was the alginate harvested from a culture of themucoid form of Pseudomonas aeruginosa which was isolated from the lungsof a patient with cystic fibrosis. This alginate of bacterial origin isa copolymer of ο-acetylated β(1-4)-D-mannuronic acid and its C5 epimerL-glucuronic acid but unlike the seaweed type alginate derived fromseaweeds such as Eisenia bicyclis, it has been highly acetylated.

[Molecular weight]

The molecular weight was determined by gel filtration chromatographyusing Sephadex G-150 (Pharmacia, Sweden). Thus, the enzyme solution wasapplied to a column of Sephadex G-150 equilibrated with 10 mM Tris- HClbuffer (pH 7.0) and the enzyme was eluted with the same buffer at 4° C.in 3.0 ml fractions every 4 minutes for molecular weight determination.The results are shown below in Table 1.

                  TABLE 1                                                         ______________________________________                                                     Al-I lyase                                                                            Al-III lyase                                             ______________________________________                                        Localization   Cytoplasm Cytoplasm                                            Molecular weight                                                                             60000     38000                                                Subunit          1         1                                                  ______________________________________                                    

[Effects of pH and temperature]

The data are presented below in Table 2.

                  TABLE 2                                                         ______________________________________                                                   Al-I lyase     Al-III lyase                                        ______________________________________                                        Optimal pH   8.0 (Tris-HCl)   Same as left                                    Stable pH    6.0-8.0          Same as left                                    Optimal temperature                                                                        70° C. (Tris-HCl,                                                                       Same as left                                    pH 7)                                                                         Stable temperature                                                                         ≦40° C. (Tris-HCl,                                                               Same as left                                    pH 7)                                                                         ______________________________________                                    

[Amino acid analysis]

The N-terminal amino acid sequence of each enzyme was determined using aprotein sequencer (tradename: Biosystem 477A, Applied Biosystems, USA)linked to an amino acid derivative analyzer (tradename: Biosystem 120APTH analyzer, Applied Biosystems, USA). The N-terminal amino acidsequences are shown below.

The N-terminal amino acid sequence of Al-I lyase:

His-Pro-Phe-Asp-Gln-Ala-Val-Val-Lys-Asp-Pro-Thr-Ala-Ser-Tyr-Val-Asp-Val-Lys-Ala-(Seq. ID NO: 1)

The N-terminal amino acid sequence of Al-III lyase:

His-Pro-Phe-Asp-Gln-Ala-Val-Val-Lys-Asp-Pro-Thr-Ala-Ser-Tyr-Val-Asp-Val-Lys-Ala-(Seq. ID NO: 2)

[Substrate specificity]

Al-I lyase: Highly capable of lysing alginate of the bacterial origin

Al-III lyase: Very highly capable of lysing alginate of the bacterialorigin

[Effect of chemicals]

The data are presented below in Table 3.

                  TABLE 3                                                         ______________________________________                                                       Al-I lyase                                                                            Al-III lyase                                           ______________________________________                                        HgCl.sub.2 (1.0 mM)                                                                            +         +                                                  CuCl.sub.2 (5.0 mM)                                                                            +         +                                                  FeCl.sub.2 (5.0 mM)                                                                            +         +                                                  Glutathione (10 mM)                                                                            -         -                                                  Dithiothreitol (10 mM)                                                                         -         -                                                  Ethylenediamine  -         -                                                  tetraacetate (10 mM)                                                          ______________________________________                                         + inhibited,                                                                  - not inhibited                                                          

The Al-I lyase and Al-III lyase for use in this invention can beproduced by cultivating Flavobacterium sp. OTC-6, as isolated from soil,in an alginate-containing medium.

Flavobacterium sp. OTC-6 is a novel microorganism never described in theliterature before and has been deposited on Feb. 15, 1993 with theNational Institute for Bioscience and Human Technology at 1-3, Higashi 1chome, Tsukuba-shi, Ibaraki-ken 305 JAPAN, under the accession number ofFERM BP-4189.

The bacteriological characteristics of this organism are shown below.

(a) Morphology:

(1) Cell shape and size: rods (0.3-0.6)×(1.0-1.2) μm

(2) Motility: nonmotile

(3) Flagellation: non-flagellated

(4) Sporogenesis: non-sporulating

(5) Gram's stain: negative

(b) Cultural characteristics:

(1) Broth agar plate culture:

After 24 hours of incubation at 30° C., round colonies 1-2 mm indiameter are formed. The colonies are solid and white.

(2) Standard agar plate culture:

After 24 hours of incubation at 30° C., round colonies 1-2 mm indiameter are formed. The colonies are solid and light yellow.

(3) Litmuth milk culture:

Incubation at 30° C. does not cause coagulation. The color isblue-purple and unchanged.

(c) Physiological characteristics

(1) Catalase: positive

(2) Oxidase: positive

(3) Urease: negative

(4) Phosphatase: negative

(5) OF test: negative

(6) VP test: negative

(7) Indole production: negative

(8) Hydrogen sulfide production: negative

(9) Production of acid from sugars:

Positive: glucose

Negative: arabinose, cellobiose, lactole, mannitol, raffinose, sucrose,xylose, glycerole, fructose, maltose, rhamnose

(10) Hydrolysis of starch: negative

(11) Hydrolysis of gelatin: negative

(12) Hydrolysis of esculin: negative

(13) Reduction of nitrate: positive

(14) pH for growth: 5.0-8.5

(15) Optimal temperature for growth: 28°-34° C.

(16) NaCl concentration for growth: 0-1%

(d) GC content of DNA: 63%

Cultivation of Flavobacterium sp. OTC-6 can be carried out in the samemanner as the culture of ordinary bacteria and is preferably performedin a liquid medium under aeration and agitation. Furthermore, in orderto cause this organism to produce the object enzyme, it is generallypreferable to carry out a mass culture of the order of ten and odd toseveral hundred liters or even more.

The culture medium is preferably added alginate as a component. Theamount of alginate is not particularly restricted but, generallyspeaking, is preferably about 0.1-2 weight % based on the total amountof the medium. Furthermore, the culture medium for use in this inventionmay contain, in addition to alginate, those sources of carbon andnitrogen and inorganic salts which are generally used in the cultivationof bacteria. Among said sources of carbon is glucose. Organicnitrogenous substances such as peptone, meat extract, corn steep liquor,yeast extract, etc. and inorganic nitrogen compounds such as ammoniumsulfate, ammonium chloride, etc. can be used as said sources ofnitrogen. The inorganic salts may for example be potassium dihydrogenphosphate, dipotassium hydrogen phosphate, magnesium sulfate and sodiumchloride.

Cultivation is carried out at a temperature of about 25°-40° C. underthe pH conditions of about 5.5-8.0 and is generally completed in about24-48 hours. As the resulting culture broth is purified by the per seknown means, there is obtained the object alginate lyase. For example, acell extract separated from the broth is purified by columnchromatography using DEAE-cellulose, Sephadex G-150, or hydroxylapatite,for instance.

In using said alginate lyase as a therapeutic medicine for cysticfibrosis, it can be formulated into various pharmaceutical compositionsusing special care not to cause inactivation of the enzyme. It isessential to avoid applying excessive heat or using a carrier tending todeactivate the enzyme. Thus, it is recommendable to use such additivesas polyethylene glycol, physiological saline, phosphate buffer, etc. inthe manufacture of a spray or solution, such additives as starch,lactose, etc. in the manufacture of tablets or powders, such additivesas olive oil, polyethylene glycol, etc. in the manufacture of anemulsion, or such additives as hard gelation etc. in the manufacture ofcapsules.

It is also within the scope of this invention to incorporate abronchodilator such as aminophylline, an antibiotic drug such as apenicillin, cephem or new quinolone, a DNase, a protease inhibitorand/or an amiloride in suitable amounts for enhanced therapeuticefficacy.

The unit dosage form of such a pharmaceutical composition of thisinvention can be chosen according to the therapeutic objective, andincludes a spray, infusion, tablet, solution, emulsion, powder, capsule,injection and so on.

There is no particular limitation on the modality of treatment with thepharmaceutical composition of this invention and the composition can beadministered according to a treatment protocol which depends on thepatient's age, sex and other factors, the severity of disease and so on.By way of illustration, a spray or an infusion is directly applied tothe affected site by spraying or infusion. The tablet, solution,emulsion, powder and capsule are administered orally. The injection isadministered intravenously, either as it is or in admixture with anordinary infusion fluid such as glucose solution, amino acid infusionand so on.

The dosage of the pharmaceutical composition of this invention can bejudiciously selected according to the method of administration, sex andother patient factors, the severity of disease, etc. but in terms of theactive ingredient alginate lyase, the daily dose per kg body weight isgenerally 0.1-100 mg and preferably 1-10 mg.

BEST MODE FOR CARRYING OUT THE INVENTION

The following reference examples, pharmacological test examples andformulation example are intended to describe the invention in furtherdetail.

Reference Example 1 (Preparation of Al-I lyase)

Flavobacterium sp. OTC-6 was inoculated into 10 l of an autoclavedliquid medium (containing 1.50% of sodium alginate, 0.10% of ammoniumsulfate, 0.05% of magnesium sulfate septahydrate, 0.10% of potassiumdihydrogen phosphate, 0.40% of disodium hydrogen phosphate 12 hydrate,and 0.05% of yeast extract; pH 7.2) and cultivated under shaking at 30°C. for 24 hours. The resulting broth was centrifuged to harvest 73 g ofcells. The cells were suspended in 60 ml of 10 mM Tris-HCl buffer (pH7.0), sonicated (9 KHz, 170 W, 10 min.) and centrifuged (25000 G, 30min.) to separate a supernatant (cell extract).

This cell extract (120 ml, protein 6.68 g) was purified using a columnof DEAE-cellulose (Wako Pure Chemical) equilibrated with 10 mM Tris-HClbuffer (pH 7.0) (4 cm×25 cm, 0-0.5M NaCl linear gradient elution).

The resulting active fraction (250 ml, protein 1.78 g) was furtherpurified using a column of hydroxylapatite (Tosoh) equilibrated with 5.0mM potassium phosphate buffer (hereinafter referred to as KPB) (pH 7.0)(4 cm×25 cm, 5-500 mM KPB linear gradient elution, pH 7.0) and theeluate was concentrated (20 ml, protein 208 mg) using a ultrafiltermembrane (Amicon PM10, Grace Japan).

The concentrate was passed through a column (2.5 cm×60 cm) of SephadexG-150 (Pharmacia, Sweden) equilibrated with 10 mM Tris-HCl buffer (pH7.0) and the active fraction (75 ml, protein 191 mg) was furtherpurified on a column of QAE-Sephadex A-25 (Pharmacia) equilibrated with10 mM Tris-HCl buffer (pH 7.0) (2.5 cm×30 cm, 0-0.5M NaCl lineargradient elution). This product was designated as Al-I lyase.

Reference Example 2 (Preparation of Al-III lyase)

Using a two-ton fermentation tank, 1400 l of an autoclaved liquid medium(containing 1.00% of sodium alginate, 0.01% of ammonium sulfate, 0.05%of magnesium sulfate septahydrate, 0.10% of potassium dihydrogenphosphate, 0.40% of disodium hydrogen phosphate 12-hydrate and 0.05% ofyeast extract; pH 7.2) was inoculated with Flavobacterium sp. OTC-6 at1.2% inoculation level and the inoculated medium was incubated at 30° C.for 17 hours. This cultural procedure was carried out under agitation(170 rpm) and air sparging at a flow rate of 200 l/min. The cells (350g) harvested from the culture broth amounting to 100 l were suspended in5 l of 5.0 mM KPB (pH 7.0) and the suspension was passed through a celldisintegrator (Dyno-Mill-KDL, Shinmaru Enterprises Corporation) andcentrifuged (25000 G, 30 min.) to separate a supernatant (cell extract).

This cell extract (5.54 l, protein 51.2 g) was passed through a column(4.6 cm×18 cm) of DEAE-Cellulofine (Seikagaku Kogyo) equilibrated with5.0 mM KPB (pH 7.0) and an active fraction was obtained as the effluent(6.05 l, protein 18.9 g).

This fraction was purified by column chromatography using a column (12cm×18 cm) of hydroxylapatite (Nacalai Tesque) equilibrated with 5.0 mMKPB (pH 7.0) by the stepwise elution method (20 mM, 40 mM and 100 mMKPB, pH 7.0).

The Al-III-containing fraction (2.64 l, protein 984 mg) eluted with 40mM KPB (pH 7.0) was saturated 40% with ammonium sulfate and purifiedusing a column of Butyl-Sepharose F.F. (Pharmacia) equilibrated with 5.0mM KPB (pH 7.0) similarly saturated 40% with ammonium sulfate (4.6 cm×10cm, 40-0% ammonium sulfate-saturated 5.0 mM KPB linear gradient elution,pH 7.0).

The resulting active fraction was concentrated (66 ml, protein 218 mg)using a ultrafilter membrane (Amicon PM10, Grace Japan) and passedthrough a column (5.0 cm×60 cm) of Sephacryl S-200 HR (Pharmacia)equilibrated with 5.0 mM KPB (pH 7.0). The resulting active fraction(176 ml, protein 153 mg) was passed through a column (4.6 cm×6.0 cm) ofS-Sepharose F. F. (Pharmacia) equilibrated with 5.0 mM KPB (pH 7.0) andlinear pH gradient elution [5.0 mM KPB (pH 7.0)-5.0 mM dipotassiumhydrogen phosphate (pH 8.6)] was carried out. The active fraction thusobtained was designated as Al-III lyase.

Reference Example 3 (Preparation of Al-II lyase)

Flavobacterium sp. OTC-6 was inoculated into 12 l of an autoclavedliquid medium (containing 0.20% of sodium alginate, 0.10% of ammoniumsulfate, 0.05% of magnesium sulfate septahydrate, 0.10% of potassiumdihydrogen phosphate, 0.40% of disodium hydrogen phosphate 12-hydrateand 0.05% of yeast extract; pH 7.2) and cultivated aerobically at 30° C.for 24 hours. The resulting broth was centrifuged to harvest 60 g ofcells.

The cells were suspended in 100 ml of 5 mM Tris-HCl buffer (pH 7.0),sonicated (9 KHz, 10 min., 0° C.) and centrifuged (25000 G, 30 min.) toseparate a supernatant (cell extract). This cell extract (160 ml,protein 4.97 g) was purified using a column of CM-cellulose equilibratedwith 10 mM Tris-HCl buffer (pH 7.0) (4.6 cm×46 cm; 0-0.6M NaCl lineargradient elution). The resulting active fraction (1235 ml, protein 608mg) was further purified with a column of hydroxylapatite (NacalaiTesque) equilibrated with 5.0 mM KPB (pH 7.0) (4.6 cm×26 cm; 5-500 mMKPB linear gradient elution; pH 7.0).

The resulting active fraction (284 ml, protein 34.8 mg) was saturated30% with ammonium sulfate and purified using a column of Butyl-Toyopearl650M (Tosoh) equilibrated with 5.0 mM KPB (pH 7.0) similarly saturated30% with ammonium sulfate (1.5 cm×15 cm, 30-0% ammoniumsulfate-saturated 5.0 mM KPB linear gradient elution; pH 7.0).

The active fraction thus obtained was concentrated (5 ml, protein 10 mg)using a ultrafilter membrane (Amicon PM10, Grace Japan), passed througha column (2.6 cm×74 cm) of Toyopearl HW-55 (Tosoh) equilibrated with 5.0mM KPB (pH 7.0), and concentrated again with a ultrafilter membrane. Theproduct was designated as Al-II lyase.

The Al-II lyase prepared as above has an N-terminal amine acid sequencecorresponding to SEQ ID NO: 3, which appears hereinafter, and has thefollowing physicochemical properties. These physicochemical propertieswere determined by the methods already described for the alginate lyasesAl-I and Al-III.

(1) Activity: This enzyme lyses alginate to saccharides having anon-reducing end C₄ -C₅ double bond and ultimately to4-deoxy-5-ketouronic-acid.

(2) Molecular weight: 25000

(3) Optimal pH: 8.0

(4) Stable pH: 6.0-8.0

(5) Optimal temperature: 70° C.

(6) Substrate specificity: The enzyme acts on alginate, showing aparticularly high lytic action on the alginate derived from Eiseniabicyclis.

Pharmacological Test Example 1

The mucoid form of Pseudomonas aeruginosa was isolated from cysticfibrosis patients by Dr. A. Prince at Colombia University. All thespecies (7 species) of this mucoid form of Ps. aeruginosa wereaerobically cultivated in 100 ml of TSB (Tryptic Soy Broth, DifcoLaboratories, USA) at 30° C. for 20 hours. The cells were removed bycentrifugation and 4 volumes of 95% ethanol was added to thesupernatant. The mixture was allowed to stand at 4° C. for 3 hours andthe sediment was centrifugally collected and washed with 95% ethanoltwice and, then, with 100% ethanol. The sediment was dried in vacuo atroom temperature to provide a bacterial alginate. This alginate wasdissolved in water and used as the substrate for the assay of alginatelyase activity.

The alginate was quantitated by the carbazole method using the sodiumalginate (molecular weight 25700) from Eisenia bicyclis as manufacturedby Sigma Chemical, USA as the standard. The DNA was assayed by thediphenylamine method using salmon testicular DNA (Sigma Chemical) as thestandard. As the alginate lyase, the Al-I lyase, Al-II lyase and Al-IIIlyase prepared in the above Reference Examples were respectively used.

The activities of these enzymes were respectively assayed by thealginate lyase assay method described hereinbefore.

The results are presented in Table 4.

                                      TABLE 4                                     __________________________________________________________________________    Cell weight *1)                                                                           DNA *2)                                                                            Alginate *3)                                                                         Specific activity (U/mg) *4)                          Strain                                                                            (g)     (mg) (mg)   Al-I lyase                                                                          Al-III lyase                                                                         Al-II lyase                              __________________________________________________________________________    1   1.22    22.3 234    68.3  125    0                                        2   1.29    30.1 228    62.1  132    0                                        3   0.83    58.4 195    59.8  109    0                                        4   1.62    32.3 50.3   62.5  128    0                                        5   1.43    80.1 155    64.3  113    0                                        6   1.21    7.8  60.1   59.8  133    0                                        7   1.32    8.3  49.2   52.5  127    0                                        A   --      --   --     63.3  132    0                                        Sodium alginate         134   30     146                                      __________________________________________________________________________

In Table 4 above, *1) denotes the weight of the moist cells harvestedfrom 100 ml of the broth, *2) denotes the amount of DNA accumulated in100 ml of the broth, *3) denotes the amount of alginate accumulated in100 ml of the broth, and *4) denotes the activity per mg of the enzymeas calculated with the amount of enzyme increasing the absorbance at 235nm by "1" in 1 minute being taken as unity (U). The symbol A in theStrain column represents the alginate harvested from a culture of themucoid form of Pseudomonas aeruginosa isolated by Dr. A. M. Chakrabarty,and sodium alginate represents the sodium salt of alginate derived fromEisenia bicyclis as manufactured by Sigma Chemical.

The following can be seen from Table 4. Thus, whereas Al-II lyase showsno activity against any of the alginates produced by said 7 species ofthe mucoid form of Ps. aerugionosa, Al-I lyase and Al-III lyase showactivity on all of these alginates and Al-III lyase, in particular, isremarkably active on the alginates. Since Al-I lyase and Al-III lyaseshow high substrate specificity for the alginates responsible for themucus in patients with cystic fibrosis as demonstrated above, they aresuggested to be effective in the treatment of cystic fibrosis.

Pharmacological Test Example 2

To the mucous substance produced by the mucoid form of Pseudomonasaeruginosa isolated from a cystic fibrosis patient was added the Al-Ilyase prepared in Reference Example 1 above and the change in viscosityof the mucus was investigated. Thus, 20 μg of Al-I lyase was added to 20ml of the mucous substance produced by Strain 1 mentioned in Table 4above (containing 46.8 mg of alginate and 4.46 mg of DNA) and theviscosity of the mucus was serially measured with a viscosimeter at 25°C. FIG. 1 diagrammatically shows the time course of viscosity of themucous substance after addition of Al-I lyase.

For comparison's sake, 20 μg each of DNase I and DNase II (SigmaChemical) were added to 20 ml of the above mucus, 20 μg of the Al-IIlyase prepared in Reference Example 3 above was added to 20 ml of thesame mucus as above and the change in viscosity of the mucous substancein each case was investigated. FIG. 2 diagrammatically shows the timecourse of viscosity of the mucous substance after addition of DNase Iand DNase II. FIG. 3 diagrammatically shows the time course of viscosityof the mucous substance when Al-II lyase was added. FIG. 4diagrammatically shows the time courses of viscosity of the mucoussubstance over a period of 60 minutes following addition of each enzyme.

It is clear from FIGS. 1 and 4 that addition of Al-I lyase causes arapid decrease in viscosity of the mucous substance. It was alsoconfirmed that by 1 hour following addition of Al-I lyase, the viscosityof the mucous substance had dropped to a level comparable to theviscosity of water. However, when any of DNase I, DNase II and Al-IIlyase was added, the viscosity of the mucus was not decreased at all.

Formulation Example

In a 150 mM sodium chloride-1 mM calcium chloride solution was dissolved4 mg/ml of the Al-III lyase prepared in Reference Example 2 above toprovide a spray preparation.

Sequence Table

Sequence Number: 1

Sequence length: 20

Sequence type: amino acids

Topology: linear

Molecule type: protein

Sequence:

His Pro Phe Asp Gln Ala Val Val Lys Asp Pro Thr Ala Ser Tyr Val Asp ValLys Ala

Sequence Number: 2

Sequence length: 20

Sequence type: amino acids

Topology: linear

Molecule type: protein

Sequence:

His Pro Phe Asp Gln Ala Val Val Lys Asp Pro Thr Ala Ser Tyr Val Asp ValLys Ala

Sequence Number: 3

Sequence length: 20

Sequence type: amino acids

Topology: linear

Molecule type: protein

Sequence:

Ala Pro Ala Ala Ala His Ser Ser Ile Asp Leu Ser Lys Xaa Lys Leu Gln IlePro Val

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the time course of viscosity of the mucoussubstance after addition of Al-I lyase. FIG. 2 is a graph showing thetime course of viscosity of the mucous substance after addition of DNaseI and DNase II. FIG. 3 is a graph showing the time course of viscosityof the mucous substance after addition of Al-II lyase. FIG. 4 is a graphshowing the changes in viscosity of the mucous substance over a periodof 60 minutes following addition of Al-I lyase, DNase (DNase I and DNaseII) and Al-II lyase, respectively.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 3                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20                                                                (B) TYPE: amino acids                                                         (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       HisProPheAspGlnAlaValValLysAspProThrAlaSerTyrVal                              151015                                                                        AspValLysAla                                                                  20                                                                            (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20                                                                (B) TYPE: amino acids                                                         (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       HisProPheAspGlnAlaValValLysAspProThrAlaSerTyrVal                              151015                                                                        AspValLysAla                                                                  20                                                                            (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20                                                                (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       AlaProAlaAlaAlaHisSerSerIleAspLeuSerLysXaaLysLeu                              151015                                                                        GlnIleProVal                                                                  20                                                                            __________________________________________________________________________

We claim:
 1. An isolated alginate lyase enzyme wherein said enzyme iscapable of lysing alginate in the mucous substance produced in a patientwith cystic fibrosis and wherein said enzyme has an N-terminal aminoacid sequence corresponding to SEQ ID No.: 1, may be obtainable fromFlavobacterium OTC-6 and has the following physicochemicalproperties:(1) Activity: lyses alginate to saccharides having anon-reducing end C₄ -C₅ double bond and ultimately to4-deoxy-5-ketouronic acid; (2) Molecular weight: 60,000; (3) Optimal pH:80; (4) Stable pH: 6.0-8.0; (5) Optimal temperature: 70° C.; (6)Substrate Specificity: alginate.
 2. An isolated alginate lyase enzymewherein said enzyme is capable of lysing alginate in the mucoussubstance produced in a patient with cystic fibrosis and wherein saidenzyme has an N-terminal amino acid sequence corresponding to SEQ IDNo.: 2, may be obtainable from Flavobacterium OTC-6 and has thefollowing physicochemical properties:(1) Activity: lyses alginate tosaccharides having a non-reducing end C₄ -C₅ double bond and ultimatelyto 4-deoxy-5-ketouronic acid; (2) Molecular weight: 38,000; (3) OptimalpH: 8.0; (4) Stable pH: 6.0-8.0; (5) Optimal temperature: 70° C.; (6)Substrate Specificity: alginate with a particularly high lytic activityon alginate of the bacterial origin.
 3. A pharmaceutical composition forthe treatment of cystic fibrosis comprising an effective amount of theenzyme of claim 1 and a pharmaceutically acceptable carrier or diluent.4. A pharmaceutical composition for the treatment of cystic fibrosiscomprising an effective amount of the enzyme of claim 2 and apharmaceutically acceptable carrier or diluent.
 5. The pharmaceuticalcomposition of claim 3 further comprising a DNase or an antibiotic. 6.The pharmaceutical composition of claim 4 further comprising a DNase oran antibiotic.
 7. The pharmaceutical composition as claimed in any oneof claims 3, 4, 5 or 6 which is provided in a unit dosage form of spray,solution or emulsion.
 8. A method of treating cystic fibrosis comprisingadministering an effective amount of the pharmaceutical composition ofclaim 7 by spraying or coating to the affected site.
 9. Thepharmaceutical composition of any one of claims 3, 4, 5 or 6, whereinthe enzyme is capable of lysing a highly acetylated alginate comparedwith an alginate derived from seaweeds, the acetylated alginate beingone produced by the microorganisms of the genus Pseudomonas, thealginate lyase being present in an effective amount capable of lysingthe alginate in mucous substance produced in a patient with cysticfibrosis.
 10. A method of treating cystic fibrosis comprisingadministering an effective amount of the enzyme of claim 1 or claim 2 byspraying or coating to the affected site.